1. Field of the Invention
The present invention relates to a method of producing a cloned animal by demecolcine treatment. More particularly, the present invention relates to a method of effectively producing a cloned animal by producing a nuclear transfer embryo of an animal treated with demecolcine after activation of a fused oocyte, culturing the nuclear transfer embryo in vitro, and transferring the nuclear transfer embryo in vivo.
2. Description of the Related Art
With the recent development of somatic cell nuclear transfer (SCNT) technology by cell fusion or intracytoplasmic cell injection, animals are actually being cloned. SCNT technology, which allows living offspring to be born without undergoing meiosis and haploid germ cell retention which generally occur in a generative process, is a method of developing new individuals by transferring diploid somatic cells of adults into enucleated cells to create embryos and then transferring the embryos in vivo.
Such SCNT technology can be widely used in the field, for example, in the propagation of superior animals, conservation of rare or nearly extinct animals, production of certain nutrients, production of therapeutic bio-materials, production of animals for organ transplantation, production of animals with diseases or disorders, and production of animals medically suitable for alternative treatments to organ transplantation such as gene therapy.
Animal cloning was first accomplished by Dr. Wilmut of the Roslin Institute, England, by taking a mammary gland cell from a six-year old female sheep, transferring the cell into an enucleated oocyte to prepare a nuclear transfer embryo, and transferring the embryo in vivo, thereby producing a cloned sheep named Dolly. Thereafter, it was reported that cloned cows, mice, goats, pigs and rabbits were produced by nuclear transfer using somatic cells (see WO 9937143A2, EP 930009A1, WO 9934669A1, WO 9901164A1 and U.S. Pat. No. 5,945,577).
However, it is very difficult to implement cloning by any of these methods due to low in vitro and in vivo development rates of the SCNT embryos. Particularly, pigs showed very low in vitro development to blastocyst, had low number of cells, and showed discontinuous development of fertilized embryos cloned since in vivo transfer. Thus, to efficiently produce a cloned pig, a novel method was needed to improve the development to blastocyst, a stage prior to the implantation of NT embryos.
Accordingly, while researching an improved method of producing a cloned animal using SCNT technology, the present inventors found that in vitro development of a nuclear transfer embryo was significantly improved by treatment with a specific chemical, demecolcine, after the fused nuclear transfer embryo was activated during the process of producing a cloned animal using SCNT technology. Also, maintenance of pregnancy from transfer of the embryos in a surrogate mother to delivery significantly improved compared to the control group. These experimental findings are the basis of the present invention.